The process of transthyretin (TTR) amyloidogenesis leads to peripheral neuropathy, organ dysfunction, and in rare cases central nervous system pathology (Sekijima, Y.; et al. Lab. Invest. 2003, 83, 409-417; Hammarström, P.; et al. Biochemistry, 2003, 42, 6656-6663; Garzuly, F.; et al. Neurology, 1996, 47, 1562-1567; Ikeda, S.; et al. Neurology, 2002, 58, 1001-1007; Westermark, P.; et al. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2843-2845; Jacobson, D. R.; et al. N. Engl. J. Med. 1997, 336, 466-473; Sipe, J. D. Crit. Rev. Clin. Lab. Sci. 1994, 31, 325-354). The disease caused by wild type (WT) TTR deposition, senile systemic amyloidosis (SSA), is a late onset cardiomyopathy affecting 10-25% of the population over age 80 (Westermark, P.; et al. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2843-2845). The remaining TTR-based amyloid diseases associated with point mutations are grouped into two broad classifications: familial amyloid cardiomyopathy (FAC) (Jacobson, D. R.; et al. N. Engl. J. Med. 1997, 336, 466-473) and familial amyloid polynueropathy (FAP) (Sipe, J. D. Crit. Rev. Clin. Lab. Sci. 1994, 31, 325-354). There are over 100 TTR mutations that cause the familial amyloidoses, the exact age of onset, tissue selectivity, and severity of which are dependent on the energetics of the specific mutation, the individual's genetic background, and possibly environmental factors (White, J. T.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13019-13024; Hammarström, P.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16427-16432).
The only treatment currently available for FAP is gene therapy mediated by surgical replacement of the patient's liver, the organ secreting TTR subject to misfolding into the blood stream (Herlenius, G.; et al. Transplantation 2004, 77, 64-71). The disadvantages of this approach include its invasiveness for both the donor and recipient, the requirement for life-long immune suppression, and the limited effectiveness for some mutations for reasons that are not yet clear (Olofsson, B.-O.; et al. Transplantation 2002, 73, 745-751). Currently there is no effective treatment for SSA associated with WT-TTR deposition. Therefore, a generally applicable, small molecule therapeutic strategy for all TTR-based amyloid diseases would be welcomed.
Interallelic trans-suppression in a compound heterozygous family enabled by the inclusion of T119M transthyretin subunits into tetramers otherwise composed of disease associated subunits (V30M), demonstrates that kinetic stabilization of TTR is sufficient to ameliorate FAP (Hammarström, P.; et al. Science 2003, 299, 713-716; Coelho, T.; et al. J. Rheumatol. 1993, 20, 179; Coelho, T.; et al. Neuromusc. Disord. 1996, 6, 27). The efficacy of trans-suppression implies that small molecule native state kinetic stabilization should also ameliorate amyloidosis (Sacchettini, J. C.; Kelly, J. W. Nat. Rev. Drug Disc. 2002, 1, 267-275; Cohen, F. E.; Kelly, J. W. Nature 2003, 426, 6968, 905-909). The utility of small molecules to tune the free energy landscape of proteins to prevent misfolding associated with disease has now been demonstrated in several instances (Hammarström, P.; et al. Science 2003, 299, 713-716; Saccheftini, J. C.; Kelly, J. W. Nat. Rev. Drug Disc. 2002, 1, 267-275; Cohen, F. E.; Kelly, J. W. Nature 2003, 426, 6968, 905-909; De Lorenzi, E.; et al. Curr. Med. Chem. 2004, 11, 1065-1084; Hardy, J.; et al. Science 2002, 297, 353-356; Ray, S. S.; Lansbury, P. T., Jr. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5701-5702; Miller, S. R.; Sekijima, Y.; Kelly, J. W. Lab. Invest. 2004, 84, 545-552).
TTR is a 127-residue β-sheet rich homotetramer characterized by 222 molecular symmetry, possessing two thyroxine (T4) binding sites (Blake, C. C.; et al. J. Mol. Biol. 1974, 88, 1-12; Blake, C. C.; et al. J. Mol. Biol. 1978, 121, 339-56). The vast majority (>99%) of the TTR T4 binding capacity in both the cerebrospinal fluid (CSF) and blood plasma is unutilized because of the high concentration of TTR and the presence of thyroid binding globulin (blood) and albumin (blood and CSF), which are also carriers of T4 (Bartalena, L.; Robbins, J. Clin. Lab. Med. 1993, 13, 583-598; Schreiber, G.; Richardson, S. J. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1997, 116, 137-160; Stockigt, J. R. Thyroid Hormone Binding and Metabolism. Endocrinology, Fourth Ed. Degroot, L. J., Jameson, J. L., Eds.; W.B. Saunders Co.: Philadelphia, 2001, Volume 2, Chapter 94, 1314-1326). Rate-limiting tetramer dissociation is required for amyloidogenesis (Colon, W.; Kelly, J. W. Biochemistry 1992, 31, 8654-8660; Hammarström, P.; et al. Science 2001, 293, 2459-2462; Lai, Z.; Colon, W.; Kelly, J. W. Biochemistry 1996, 35, 6470-6482; Lashuel, H. A.; Lai, Z.; Kelly, J. W. Biochemistry 1998, 37, 17851-17864), but is not sufficient (Jiang, X.; et al. Biochemistry 2001, 40, 11442-11452), as the resulting folded monomer must also undergo partial denaturation to misassemble (Colon, W.; Kelly, J. W. Biochemistry 1992, 31, 8654-8660; Lai, Z.; Colon, W.; Kelly, J. W. Biochemistry 1996, 35, 6470-6482; Lashuel, H. A.; Lai, Z.; Kelly, J. W. Biochemistry 1998, 37, 17851-17864; Jiang, X.; et al. Biochemistry 2001, 40, 11442-11452; Liu, K.; et al. Nat. Struct. Biol. 2000, 7, 754-757). Previous studies demonstrate that T4 binding inhibits TTR aggregation by kinetic stabilization of the native state. The activation barrier for dissociation is increased by preferential stabilization of the native tetramer relative to the dissociative transition state (Hammarström, P.; et al. Science 2003, 299, 713-716; Miroy, G. J.; et al. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15051-15056; Peterson, S. A.; et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12956-12960).
Screening, structure-based design, and lead compound optimization by parallel synthesis has led to several other structurally distinct classes of potent TTR amyloidogenesis inhibitors (Hammarström, P.; et al. Science 2003, 299, 713-716; Sacchettini, J. C.; Kelly, J. W. Nat. Rev. Drug Disc. 2002, 1, 267-275; Cohen, F. E.; Kelly, J. W. Nature 2003, 426, 6968, 905-909; Miller, S. R.; Sekijima, Y.; Kelly, J. W. Lab. Invest. 2004, 84, 545-552; Miroy, G. J.; et al. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15051-15056; Peterson, S. A.; et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12956-12960; Petrassi, H. M.; et al. J. Am. Chem. Soc. Submitted; Purkey, H. E.; et al. Chemistry & Biology. In press; Adamski-Werner, S. L.; et al. J. Med. Chem. 2004, 47, 355-374; Green, N. S.; et al. J. Am. Chem. Soc. 2003, 125, 13404-13414; Petrassi, H. M.; et al. J. Am. Chem. Soc. 2000, 122, 2178-2192; Baures, P. W.; et al. Bioorg. Med. Chem. 1998, 6, 1389-1401; Oza, V. B.; et al. Bioorg. Med. Chem. Lett. 1999, 9, 1-6; Baures, P. W.; et al. Bioorg. Med. Chem. 1999, 7, 1339-1347; Klabunde, T.; et al. Nat. Struct. Biol. 2000, 7, 312-321; Oza, V. B.; et al. J. Med. Chem. 2002, 45, 321-332; Razavi, H.; et al. Angew. Chem. Int. Ed. 2003, 42, 2758-2761). Effective inhibitors generally have two aryls linked directly or through a spacer such as an amine, an ether, or an ethylene bridge. Optimally, one aryl is functionalized with halogens or aliphatic groups (typically occupying the inner cavity of the thyroxine binding site), and the other by a hydroxyl and/or carboxylic acid (which can interact electrostatically with the Lys-15 e-NH3+ and/or Glu-54 carboxyl groups at the periphery of the outer binding cavity) (Hammarström, P.; et al. Science 2003, 299, 713-716; Sacchettini, J. C.; Kelly, J. W. Nat. Rev. Drug Disc. 2002, 1, 267-275; Cohen, F. E.; Kelly, J. W. Nature 2003, 426, 6968, 905-909; Miller, S. R.; Sekijima, Y.; Kelly, J. W. Lab. Invest. 2004, 84, 545-552; Miroy, G. J.; et al. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15051-15056; Peterson, S. A.; et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12956-12960; Petrassi, H. M.; et al. J. Am. Chem. Soc. Submitted; Purkey, H. E.; et al. Chemistry & Biology. In press; Adamski-Werner, S. L.; et al. J. Med. Chem. 2004, 47, 355-374; Green, N. S.; et al. J. Am. Chem. Soc. 2003, 125, 13404-13414; Petrassi, H. M.; et al. J. Am. Chem. Soc. 2000, 122, 2178-2192; Baures, P. W.; et al. Bioorg. Med. Chem. 1998, 6, 1389-1401; Oza, V. B.; et al. Bioorg. Med. Chem. Lett. 1999, 9, 1-6; Baures, P. W.; et al. Bioorg. Med. Chem. 1999, 7, 1339-1347; Klabunde, T.; et al. Nat. Struct. Biol. 2000, 7, 312-321; Oza, V. B.; et al. J. Med. Chem. 2002, 45, 321-332; Razavi, H.; et al. Angew. Chem. Int. Ed. 2003, 42, 2758-2761). Both cavities have hydrophobic depressions called halogen binding pockets, that are complemented by the aryl substructures and their hydrophobic substituents.
After considering the orientation and placement of substituted aromatics in numerous TTR.(inhibitor)2 co-crystal structures, synthetic accessibility, and the potential for future high-throughput dynamic combinatorial library analyses (Nazarpack-Kandlousy, N.; et al. J. Comb. Chem. 1999, 199-206; Hochgürtel, M.; et al. Proc. Nat. Acad. Sci., U.S.A. 2002, 99, 3382-3387), we chose to explore the aldoxime ether moiety to link the two aryl rings. There are several FDA approved antibacterial agents containing the oxime ether moiety, suggesting that this substructure is compatible with human biology (FDA approved antibacterial agents containing the oxime ether moiety were found using MDL ISIS/Base 2.5, from MDL Information Systems, Inc., MDDR 2003.2 (25.11) database, which scans the Drug Data Report from Prous Science Publishers containing data regarding the development of pharmaceuticals.). The goal of this study is to find bisarylaldoxime ether structures that bind with high affinity to TTR in human blood plasma (Purkey, H. E.; et al. Proc. Natl. Acad. Sci. U.S.A 2001, 98, 5566-5571) and stabilize the native state against amyloidogenesis (Hammarström, P.; et al. Science 2003, 299, 713-716; Cohen, F. E.; Kelly, J. W. Nature 2003, 426, no. 6968, 905-909).